Project Summary The cytoskeleton is essential to many aspects of life, cell division and motility being prime examples. The overarching goal of this proposal is to understand the roles of two actin nucleators, Spire (Spir) and Cappuccino (Capu). We are studying these protein in Drosophila, where they are known to be essential to polarity establishment during early development. Spir and Capu build an actin mesh that traverses the Drosophila oocyte throughout mid-oogenesis. The presence of the mesh and its removal at the correct developmental stage are both critical to polarity establishment. Thus learning how Spir and Capu collaborate to build the mesh is essential to a fundamental understanding of how they contribute to polarity establishment during development. In the proposed work, we will focus on two aspects of Spir-Capu operation: 1) the mechanism by which they assemble actin filaments and 2) how Spir and Capu are spatially and temporally regulated, in order to establish the actin mesh and then remove it when appropriate. We have developed a working model in which Spir and Capu are both active throughout mid-oogenesis (stages 5-9); Spir enhances nucleation by Capu, specifically at membranes; together they build a dynamic actin mesh until stage 10 when Spir and Capu protein levels drop, the mesh disappears, and streaming begins. We will test this model using both in vitro and in vivo approaches with assays that span scales from single molecule fluorescence microscopy to whole animal genetics. Testing this model will lead to a mechanistic understanding of two proteins, conserved throughout metazoan species. It will advance our knowledge of the cytoskeleton and how it is controlled. Given the co-existence of this pair of proteins in polar cells, including neurons and epithelial cells in mammals, it has been proposed that their role as polarity factors in Drosophila is conserved. Thus we anticipate that what is learned about Spir and Capu in Drosophila oogenesis will be applicable to our understanding of the cytoskeleton, cell polarity, fertility, development and health in many animals, ranging from Drosophila to humans.